1. Field of the Invention
The present invention relates to fluorogenic derivatives of 2,1,3-benzoxadiazoles and more specifically to compounds of the general formula: ##STR3## wherein X is ##STR4## --CN,--CO.sub.2 CH.sub.3 or SO.sub.2 R, and wherein R is ##STR5## OR.sub.3, or --Cl; R.sub.1 and R.sub.2 are hydrogen atoms or alkyl groups 1 to 3 carbon atoms, and R.sub.3 is a phenyl or benzyl group. The invention also relates to a method of detecting and quantifying thiols as well as primary and secondary amines by reacting them with the above 2,1,3-benzoxadiazole derivatives to form a novel fluorescent product.
2. Description of the Prior Art
Assaying techniques wherein a fluorogenic reagent is reacted with a substrate to form a fluorescing adduct have been known for some time. Such techniques have been found to be particularly well-suited to biological assaying. Thus, in U.S. Pat. No. 4,045,487, a furanone compound is disclosed which, although non-fluorescent by itself, forms a fluorescent product when reacted with a primary amine. Although the fluorescent furanone complexes are readily detectable and quantifiable by conventional techniques, their practical application to biological media to be analyzed is limited due to the non-reactivity of the furanones with secondary amines and thiols. Thus, unless there is interest only in the detection and measurement of particular primary amines, other time consuming and potentially expensive techniques will be required in order to assay secondary amines as well as thiols.
The use of fluorogenic reagents has been found to increase the sensitivity of recently developed techniques in high performance liquid chromatography (HPLC), which enables more efficient separation of amino acids and thiols from biological fluids. While fluorogenic reagents such as fluorescamine and o-phthalaldehyde are well suited for the sensitive detection of amino acids having primary amino groups, they, like the furanone compounds described above, are not reactive with the imino acids (secondary amines) such as proline and hydroxyproline, and therefore do not form fluorescent complexes therewith. Thus, in order to assay imino acids with the above fluorogenic reagents, it is required that oxidizing reagents such as N-chlorosuccinimide or sodium hypochlorite be added first so as to convert the imino acids to primary amines. Such is not desirable in practice however since the presence of excess oxidizing reagents obstructs the fluorescence yield of the generated fluorophores.
The deleterious side reactions of the oxidizing agents as well as the undesirability of having to carry out the additional oxidation step led to the pursuit of compounds which would be capable of reacting directly with both primary and secondary amines and, optimally, thiols since such represent a significant portion of the compounds found in many biological solutions. To that end, derivatives of 2,1,3-benzoxadiazoles and more specifically, 7-fluoro-4-nitrobenzo-2,1,3-oxadiazoles (NBD-F) of the formula: ##STR6## were developed. These compounds were observed to react with both primary and secondary amines as well as thiols. The reaction with secondary amines can be represented by the following: ##STR7## Thus, NBD-F reacts with primary amino acids such as asparagine, glutamic acid, serine, glycine, threonine and alanine as well as the secondary amino acids proline and hydroxyproline at a pH of 8. However, tryptophan, which is also a secondary amino acid cannot be assayed with NBD-F. NBD-F can also be used to selectively assay certain thiol compounds by modifying the reaction conditions used for amino and imino acid analysis. However, the degree of fluorescence of the products formed with the --SH bond is lower than that obtained with the --NH bond of primary and secondary amino compounds.
Although, as indicated, NBD-F is reactive with primary amines, certain secondary amines and certain thiols, it has not proven completely satisfactory for use as a fluorogenic reagent. More specifically, the fluorescent adduct formed from NBD-F and thiols is very unstable due to the S.fwdarw.N migration which occurs. Additionally, the sulfur adduct has a much lower fluorescent quantum yield making the detection thereof more difficult. Finally, NBD-F itself undergoes rapid hydrolysis thereby resulting in the formation of an additional peak in the chromatogram in addition to the plurality of peaks already present for the amino acid and thiol adducts. This additional peak is to be avoided since it potentially can interfere with the peaks of analytes thus making the analytical process that much more difficult.
Ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F) as well as ammonium 7-chloro-2,1,3-benzoxadiazole-4-sulfonate (SBD-Cl) have also been employed for pre-column labelling of thiols in HPLC. SBD-Cl is a desirable fluorogenic reagent from the standpoint of water solubility as well as the stability of the SBD-thiol complex formed. Nevertheless, SBD-Cl has not proven totally satisfactory in thiol assaying techniques due to its relatively low reactivity with respect to thiol. More specifically, when SBD-Cl and thiol were reacted in an alkaline solution (pH=10) at 40.degree. C. for one hour, and then at room temperature for 24 hours, only a few percent of the SBD-Cl had reacted with thiol. It was therefore impossible to ascertain the quantity of thiol present when in low concentrations.
SBD-F, like SBD-Cl, is highly soluble in water and forms highly stable fluorophores with the thiols. It also has a greater degree of sensitivity than does SBD-Cl and excellent fluorescent characteristics. However, the conditions necessary for completion of the reaction between the thiols and SBD-F are rather drastic, with the thiols being reacted with 100 fold excess reagent in an alkaline medium (pH 9.5) at 60.degree. C. for 1 hour. Such drastic conditions should, if possible, be avoided due to the possible degradation of the thiols being analyzed.